Arginine is an important amino acid in mammals including human, which is involved in various physiological processes, including cell proliferation and growth. For example, arginine is an immediate precursor for the synthesis of the potential signal molecule nitric oxide (NO). NO functions as a neurotransmitter, a muscle relaxant or a vasodilator. The biosynthesis of NO involves nitric oxide synthase catalyzed Ca++ and NADPH-dependent reactions. Another function of arginine is as a precursor for polyamine, spermidine or spermine and participates in different physiological processes.
Studies showed that when arginine is less than 8 μM, cancer cells undergo irreversible death (Srorr & Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br. J. Cancer 30, 50). By studying the effect on cell growth, it was found that upon removal of arginine, the normal cells in the cell cycle G0 phase would enter a resting state and remain viable for several weeks without significant damage; when the concentration of arginine returned to normal level, the cells would return to normal cell cycle. However certain tumor cells would proceed from the ‘R’ point of the cell cycle G1 phase to S phase at deprivation of arginine, and undergo apoptosis soon. The apoptosis of tumor cells as a result of arginine deficiency is irreversible. Therefore, scientists began to consider treating cancers by controlling the level of arginine in the body, especially for auxotrophic tumors such as liver cancer and melanoma. This approach has now been used to study the inhibition of various tumors, including breast cancer, small cell lung cancer, prostate cancer, lymphoma and leukemia.
Arginase is the enzyme catalyzing the hydrolysis of L-arginine into ornithine and urea. In general, arginase is expressed in liver, kidney and testis of the urea-producing animals (mammals, elasmobranchs, amphibians, and turtles) as one of the enzymes of the urea cycle. Arginase catalyzes the final step of the urea recycle pathway in mammals, converting arginine to ornithine and urea. In most mammals including human, the family of arginase includes arginase I and arginase II. Arginase I is mainly expressed in the liver cells, and arginase II is mainly expressed in the kidney and erythrocytes.
There are two methods to produce arginase; of which one is to separate it from the arginase producing organism, and the other is through recombinant genetic engineering techniques. The latter has its advantages. For example, experiments have showed that a great amount of arginase could be produced by E. coli. However, there may be technical problems to produce arginase through recombinant genetic engineering techniques, such as low enzyme activity or poor stability and short half-life in vivo, limiting its application in clinical practice.
U.S. Pat. No. 7,951,366B2 disclosed a pharmaceutical composition and method for the treatment of human malignant tumor by arginine depletion using recombinant human arginase I with his-tag, wherein the arginase is modified by covalently conjugated with polyethylene glycol of molecular weight of 5,000 (MW5, 000) at the N-terminal or the amine group on the surface of the arginase. The modified human arginase had an increased stability with a half-life of 3 days in human serum.
US20100247508A1 disclosed a modified human arginase with his-tag, wherein the 168 and 303 cysteines are replaced with serines and the arginase is pegylated with polyethylene glycol of a molecular weight of 20 KDa. Like the recombinant human arginase I disclosed in U.S. Pat. No. 7,951,366B2, an additional peptide fragment, His-tag is included in the amino acid sequence of the arginase. The drug regulatory agencies in most countries do not recommend the use of these peptide fragments. For example, China State Food and Drug Administration indicates in the “Technical Quidelines on the Quality Control of Recombinant DNA Product for Human Use” that additional peptide fragments such as His-tag introduced for the purpose of simplifying the production process should be removed as far as possible from the final product.
WO 2011/008495A2 disclosed a site-directed modified arginase, in which the three cysteines were retained and another cysteine residue is introduced substituting the third amino acid residue of the N-terminus, and then pegylated with methoxy polyethylene glycol maleimide of molecular weight of 20 KDa. Since the said human arginase still carries the original three cysteine residues, its pegylation product is prone to heterogeneous and low in yield, which also adds difficulties for purification.
The present technical field has the need for a better arginase or derivatives thereof, for the treatment of arginine related diseases or disorders.